(a) Field of the Invention
The invention relates to a promoter for regulating expression of foreign genes in a transgenic organism, more specifically in a leaf-specific manner in transgenic plants.
(b) Description of Prior Art
Genetic transformation of microbes have been used for more than 15 years to produce useful recombinant molecules, and applications in the pharmaceutical, cosmaceutical and dermaceutical industries are being currently exploited. This technology has expanded from microbes to plants and animals in the last ten years with the development of techniques required to adapt this general concept to complex eukaryotic organisms. Basically a gene encoding for a protein of interest or a gene encoding for an enzyme responsible for a modification of a metabolic pathway that leads to a molecule of interest, is linked in an appropriate fashion to cis- and trans-acting regulatory sequences, and transferred to a target cell where it is incorporated in the molecular machinery (in a transitory or stable fashion). The transgenic cell, or a tissue or organism regenerated from the transgenic cell will then perform transcription and translation of the transgene and therefore be enabled to accumulate the protein of interest or to perform the new metabolic reaction through the activity of the enzyme of interest.
The emerging industry of molecular farming is one of the most promising industry of the coming century. Its promise is to provide safe and renewable molecule factories for the industry. Among the applications that are currently developed are the production of low-cost monoclonal antibodies for therapeutic and diagnostic uses, the production of unlimited amounts of hormones, cytokines and other bio-active molecules for the treatment of chronicle or lethal diseases, the production of bio-safe substitutes for various blood components, the production of unlimited amounts of processing enzymes for the food and pulp industry, the production of low-cost enzymes for waste treatments, and the production of safe bio-active molecules for the cosmetic industry.
Limitations to the application of this technology has often come from the inability of transgenic organisms to accumulate adequate amounts of the recombinant product, as a result of low transcription rates, improper splicing of the messenger, instability of the foreign mRNA, poor translation rates, hyper-susceptibility of the recombinant protein to the action of endogenous proteases or hyper-susceptibility of the recombinant organism to the foreign protein which result in improper and limited growth or in the worst cases, in strong deleterious effects to the host organism. Inadequacy of production level has a direct impact on the development of applications when profit margins are narrow, or when treatment and/or disposal of residual matter causes bio-safety or environmental problems. Improvement of the accumulation level of the desired recombinant product thus appears to be one critical factor that warrants commercialization of many applications of molecular farming.
Photosynthesis is a metabolic reaction of paramount importance in the living world. It is performed by most land plants and algae, and by some bacteria. This overall reaction involves a complex assembly of electron transfer proteins spatially arranged within the thylakoid membrane system located in the chloroplast of leaf cells. This electron transport chain is coupled at one end with the photosynthetic antennae, which comprise a variety of macro-molecules, including one molecule common to all photosynthetic organism, chlorophyll, and at the other end, to the enzymes involved in NADPH and ATP synthesis, and to the Calvin cycle, involved in coupling the release of energy from NADPH and ATP with the fixation of gaseous carbon dioxide into organic molecules. Among the proteins involved in the overall photosynthetic reaction, one, Ribulose biphosphate carboxylase (Rubisco), is the most abundant protein on earth.
Photosynthesis is thus what leaf cells are dedicated to perform, and there is an obvious interest to use promoters of genes involved in such prominent tissue-specific metabolic activity when building strong leaf-specific expression cassettes for applications in plant biotechnology.
Many of the peptidic constituents of the photosynthetic apparatus are encoded by genes present in the chloroplastic genome; as an example, the heavy subunit of Rubisco, which bears the catalytic sites for CO2 fixation, is encoded by a chloroplastic gene. However, the small subunit of this enzyme is encoded by a nuclear gene, and thus the Rubisco holo-protein is made of subunits encoded by two different genomes. For obvious reasons, there has been a great interest in trying to use Rubisco promoters to control transcription of transgenes in leaves of transgenic plants. The promoter has been extensively characterized and its use in expression vectors is protected by U.S. Pat. No. 4,962,028.
It would be highly desirable to be provided with a promoter for regulating expression of foreign genes in a transgenic organism, more specifically in transgenic plants.